Effects of Exogenous Calcium Supplementation in Water on Molting Cycle of Macrobrachium rosenbergii
DU Ting-Ting1, DING Li1, PAN Xi-Fang1, TANG Qiong-Ying1, XIA Zheng-Long2, YANG Guo-Liang1,2, YI Shao-Kui1,*
1 College of Life Sciences, Huzhou University/Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development, Huzhou 313000, China; 2 Jiangsu Shufeng Prawn Breeding Co., Ltd., Gaoyou 225654, China
Abstract：Calcium plays an important role in the molting pathway in crustaceans, and it is also an essential element for crustaceans in the biomineralization of the new epidermis in the late ecdystic period. To investigate the effects of exogenous calcium supplementation in water on molting of Macrobrachium rosenbergii, this study conducted the culture experiment with the Ca2+ concentration of 25 (control group), 85, 145 and 205 mg/L in the water. At the end of the culture experiment, the molting rate of the 4 groups was calculated. The content of Ca2+ in the hemolymph was detected, and the effects of exogenous calcium supplementation on calcium absorption and exoskeleton mineralization were investigated using scanning electron microscopy. Meanwhile, the CDS of calmodulin (MrCaM, GenBank No. OQ411017) and calreticulin (MrCRT, GenBank No. OQ411018) in the molting pathway of M. rosenbergii were obtained. The expression levels of MrCaM and MrCRT mRNA in different molting stages of M. rosenbergii were analyzed by qPCR. The results showed that the molting rate of M. rosenbergii was the highest in the 145 mg/L group, and that in 205 mg/L group was significantly lower than that in 25 mg/L group (control group)(P<0.05). In the different molting stages, calcium content in hemolymph of M. rosenbergii firstly increased and then decreased with the increase of water Ca2+ concentration, reaching the peak in the 145 mg/L group. Scanning electron microscope (SEM) results showed that exogenous calcium supplementation could promote the exoskeletal mineralization. The expression levels of MrCaM and MrCRT at different molting stages were the highest in the 145 mg/L group (P<0.05) and significantly lower in 205 mg/L group compared to the 145 mg/L group (P<0.05). The correlation analysis of MrCaM and MrCRT with hemolymph calcium ion content in the different molting stages showed that the expression levels of MrCaM were significantly correlated with hemolymph calcium ion content in molting stage (E stage)(r=0.8854), and the expression level of MrCRT was positively correlated with hemolymph calcium ion content in the different molting stages (P<0.01). Overall, the increase of Ca2+ concentration in water could promote the molting and exoskeleton mineralization of M. rosenbergii. This study provides a basis for clarifying the effects of exogenous calcium supplementation on molting in M. rosenbergii.
都婷婷, 丁丽, 潘玺方, 唐琼英, 夏正龙, 杨国梁, 易少奎. 水体外源钙补充对罗氏沼虾蜕皮周期的影响[J]. 农业生物技术学报, 2023, 31(12): 2568-2579.
DU Ting-Ting, DING Li, PAN Xi-Fang, TANG Qiong-Ying, XIA Zheng-Long, YANG Guo-Liang, YI Shao-Kui. Effects of Exogenous Calcium Supplementation in Water on Molting Cycle of Macrobrachium rosenbergii. 农业生物技术学报, 2023, 31(12): 2568-2579.
[1] 陈树桥, 陈勇, 周国勤, 等. 2012. 蜕皮激素对克氏原螯虾蜕皮和生长的影响[J]. 南京师大学报(自然科学版), 35(01): 80-83. (Chen S Q, Chen Y, Zhou G Q, et al.2012. Effects of ecdysterone on the molt and growth of Procambarus clarkii[J]. Journal of Nanjing Normal University (Natural Science Edition), 35(01): 80-83.) [2] 董双林, 堵南山, 赖伟. 1994. 日本沼虾生理生态学研究Ⅰ. 温度和体重对其代谢的影响[J]. 海洋与湖沼, 25(3): 233-237. (Dong S L, Du N S, Lai W.1994. Studies on the physio-ecology of Macrobrachium nipponenseⅠ. effects of temperature and body weight on metabolism[J]. Oceanologia Et Limnologia Sinica, 25(3): 233-237.) [3] 黄根东, 王兴仿, 倪成男, 等. 2013. 补钙在水产养殖中的应用[J]. 科学养鱼, (12): 87. (Huang G D, Wang X F, Ni C N, et al. 2013. Application of calcium supplement in aquaculture[J]. Scientific Fish Farming, (12): 87.) [4] 黎兰诗, 戴习林. 2022. 盐度对不同蜕皮时期罗氏沼虾生理生化及蜕皮相关基因表达的影响[J]. 南方农业学报, 53(8): 2302-2311. (Li L S, Dai X L.2022. Effects of salinity on Macrobrachium rosenbergii physiology, biochemistry and gene expression related to molting at different molting stages[J]. Journal of Southern Agriculture, 53(8): 2302-2311.) [5] 李英. 2010. 环境因子变化对凡纳滨对虾蜕皮同步性和生理特征影响的实验研究[D]. 硕士学位论文, 中国海洋大学, 导师: 王芳. pp. 34. (Li Y.2010. Effects of rhythm influctation of environmental factors on molting simultaneous and physiological response of Litopenaeus Vannamei[D]. Thesis of M.S. Ocean University of China, Supervisor: Wang F. pp. 34.) [6] 刘少华. 2015. EDTA滴定法测定自来水中钙镁离子含量[J]. 科技致富向导, (14): 2. (Liu S H. 2015. Determination of calcium and magnesium ion content in tap water by EDTA titration[J] Guide to Becoming Rich Through Technology, (14): 2.) [7] 卢徐斌, 姜群, 闵悦, 等. 2018. 罗氏沼虾蜕皮周期的划分及蜕皮频率对生长的影响[J]. 淡水渔业, 48(6): 88-93. (Lu X B, Jiang Q, Min Y, et al.2018. Molt staging and the effect of molting frequency on growth of Macrobrachium rosenbergii[J]. Freshwater Fisheries, 48(6): 88-93.) [8] 农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2022. 2022中国渔业统计年鉴[M]. 中国农业出版社, 北京. pp. 24. (Fishery and Fishery Administration of the Ministry of Agriculture and Rural Affairs, National Aquatic Technology Promotion Station, 2022. China Fisheries Association 2022 China Fishery Statistics Yearbook[M]. China Agricultural Publishing House, Beijing. pp. 24.) [9] 农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2021. 2021中国渔业统计年鉴[M]. 中国农业出版社, 北京. pp. 24. (Fishery and Fishery Administration of the Ministry of Agriculture and Rural Affairs, National Aquatic Technology Promotion Station, 2021. China Fisheries Association 2022 China Fishery Statistics Yearbook[M]. China Agricultural Publishing House, Beijing. pp. 24.) [10] 王慧, 房文红, 来琦芳. 2000. 水环境中Ca2+、Mg2+对中国对虾生存及生长的影响[J]. 中国水产科学, 7(1): 82-86. (Wang H, Fang W H, Lai Q F.2000. Effects of concentrations of Ca2+ and Mg2+ on survival and growth of Penaeus chinensis[J]. Journal of Fishery Sciences of China, 7(1): 82-86.) [11] 谢达祥, 陈晓汉, 黄均, 等. 2007. 水体中钙和镁对凡纳滨对虾幼体成活率和生长的影响[J]. 水利渔业, 27(5): 46-51. (Xie D X, Chen X H, Huang J, et al.2007. Effects of calcium and magnesium in water on survival rate and growth of Litopenaeus vannamei larvae[J] Water Conservancy and Fisheries, 27(5): 46-51.) [12] 徐宾朋. 2016. 拟穴青蟹蜕皮周期中离子转运相关基因的研究[D]. 博士学位论文, 浙江大学, 导师: 邵庆军. pp. 61-62. (Xu B P.2016. The functional study of the ions transport related molecules in Scvlla paramamosain during the molt cycle[D]. Thesis of PhD., Zhejiang University, Supervisor: Shao Q J. pp. 61-62.) [13] 叶成凯, 卢志杰, SARATH B V, 等. 2019. 罗氏沼虾几丁质酶3B基因的克隆及其在蜕皮周期中的表达[J]. 水产学报, 43(4): 751-762. (Ye C K, Lu Z J, Sarath B V, et al.2019. Cloning and expression analysis of chitinase-3B from giant freshwater prawn (Macrobrachium rosenbergii) during molting cycle[J]. Journal of Fisheries of China, 43(4): 751-762.) [14] 张龙涛, 吕建建, 高保全, 等. 2015. 三疣梭子蟹钙调蛋白基因的克隆及在蜕皮中的功能分析[J]. 中国水产科学, 22(6): 1150-1159. (Zhang L T, Lv J J, Gao B Q, et al.2015. Cloning and expression analysis of Portunus trituberculatus calmodulin cDNA[J]. Journal of Fishery Sciences of China , 22(6): 1150-1159.) [15] 朱长波, 董双林, 王芳. 2010. 水环境Mg2+、Ca2+含量对凡纳滨对虾幼虾生长和能量收支的影响及其机制[J]. 水产学报, 34(01): 89-96. (Zhu C B, Dong S L, Wang F.2010. Effects and mechanism of ambient Mg2+ and Ca2+ concentrations on growth and energy budget of juvenile Litopenaeus vannamei[J]. Journal of Fisheries of China, 34(01): 89-96.) [16] Cha W H, Kim Y, Lee D W.2015. Calreticulin in Cotesia plutellae suppresses immune response of Plutella xylostella (L.)[J]. Journal of Asia-Pacific Entomology, 18(1): 27-31. [17] Cheung W Y.1980. Calmodulin plays a pivotal role in cellular regulation[J]. Science, 207(4426): 19-27. [18] Chung J S, Webster S G.2006. Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas: A possible osmoregulatory role[J]. General and Comparative Endocrinology, 147(2): 206-213. [19] Cripps M C, Nakamura R M.1979. Inhibition of growth of Macrobrachium rosenbergii by calcium carbonate water hardness[C]. Proceedings of the World Mariculture Society. Oxford, UK: Blackwell Publishing Ltd, 10(1-4): 575-580. [20] Dall W.1981. Osmoregulatory ability and juvenile habitat preference in some penaeid prawns[J]. Journal of Experimental Marine Biology and Ecology, 54(1): 55-64. [21] Das S, Pitts N L, Mudron M R, et al.2016. Transcriptome analysis of the molting gland (Y-organ) from the blackback land crab, Gecarcinus lateralis[J]. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 17: 26-40. [22] Fanjul-Moles M L.2006. Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: Review and update[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 142(3): 390-400. [23] Gao Y, Gillen C M, Wheatly M G.2009. Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 152(3): 216-225. [24] Hoeflich K P, Ikura M.2002. Calmodulin in action: Diversity in target recognition and activation mechanisms[J]. Cell, 108(6): 739-742. [25] Huang H, Huang C, Guo L, et al.2019. Profiles of calreticulin and Ca2+ concentration under low temperature and salinity stress in the mud crab, Scylla paramamosain[J]. PLOS ONE, 14(7): e0220405. [26] Juneta-Nor A S, Noordin N M, Azra M N, et al.2020. Amino acid compounds released by the giant freshwater prawn Macrobrachium rosenbergii during ecdysis: A factor attracting cannibalistic behaviour[J]. Journal of Zhejiang University-Science B, 21(10): 823-834. [27] Johnson S, Michalak M, Opas M, et al.2001. The ins and outs of calreticulin: from the ER lumen to the extracellular space[J]. Trends in Cell Biology, 11(3): 122-129. [28] Lee K J, Kim H W, Gomez A M, et al.2007. Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: Cloning, tissue expression, and expression of biologically active recombinant peptide in yeast[J]. General and Comparative Endocrinology, 150(3): 505-513. [29] Lenartowski R, Suwińska A, Lenartowska M.2015. Calreticulin expression in relation to exchangeable Ca2+ level that changes dynamically during anthesis, progamic phase, and double fertilization in Petunia[J]. Planta, 241(1): 209-227. [30] Li C H, Cheng S Y.2012. Variation of calcium levels in the tissues and hemolymph of Litopenaeus vannamei at various molting stages and salinities[J]. Journal of Crustacean Biology, 32(1): 101-108. [31] Li S, Xie L, Zhang C, et al.2004. Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata)[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 138(3): 235-243. [32] Liang H, Liu Y, Zhou T T, et al.2019. Molecular characterization, RNA interference and recombinant protein approach to study the function of the putative Molt Inhibiting Hormone (FmMIH1) gene from the shrimp Fenneropenaeus merguiensis[J]. Peptides, 122: 169854. [33] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. methods, 25(4): 402-408. [34] Luana W, Li F, Wang B, et al.2007. Molecular characteristics and expression analysis of calreticulin in Chinese shrimp Fenneropenaeus chinensis[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 147(3): 482-491. [35] Means A R, Dedman J R.1980. Calmodulin-an intracellular calcium receptor[J]. Nature, 285(5760): 73-77. [36] Michalak M, Corbett e F, Mesaeli N, et al.1999. Calreticulin: One protein, one gene, many functions[J]. Biochemical Journal, 344(2): 281-292. [37] Nakamura K, Robertson M, Liu G, et al.2001. Complete heart block and sudden death in mice overexpressing calreticulin[J]. The Journal of clinical investigation, 107(10): 1245-1253. [38] Perry H, Trigg C, Larsen K, et al.2001. Calcium concentration in seawater and exoskeletal calcification in the blue crab, Callinectes sapidus[J]. Aquaculture, 198(3-4): 197-208. [39] Sakamoto K, Honto W, Iguchi M, et al.2009. Post-molt processes of cuticle formation and calcification in the Japanese mitten crab Eriocheir japonicus[J]. Fisheries Science, 75: 91-98. [40] Shen H, Hu Y, Zhang Y, et al.2014. Calcium-calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting[J]. Gene, 538(2): 235-243. [41] Spaziani E, Mattson M P, Wang W L, et al.1999. Signaling pathways for ecdysteroid hormone synthesis in crustacean Y-organs[J]. American Zoologist, 39(3): 496-512. [42] Visudtiphole V, Watthanasurorot A, Klinbunga S, et al.2010. Molecular characterization of Calreticulin: A biomarker for temperature stress responses of the giant tiger shrimp Penaeus monodon[J]. Aquaculture, 308: S100-S108. [43] Winkler A.1986. Effects of inorganic sea water constituents on branchial Na-K-ATPase activity in the shore crab Carcinus maenas[J]. Marine Biology, 92(4): 537-544. [44] Webster S G, Keller R, Dircksen H.2012. The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction[J]. General and Comparative Endocrinology, 175(2): 217-233.
